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BIOL3014 Molecular Cell Biology > Joern > Flashcards

Joern Flashcards

1
Q

What are the primary goals in studying molecular recognition in cell adhesion?

A

To understand:
The molecular mechanisms of attaching cells to the extracellular matrix (ECM).
Mechanisms of bi-directional signal transduction: inside-out and outside-in signaling.

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2
Q

What are the protein components of the ECM?

A

Collagens
Fibronectin
Tenascin
Laminin
Fibrillin

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3
Q

What polysaccharides (GAGs) are found in the ECM?

A

Hyaluronan
Dermatan sulfate
Heparin

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4
Q

Name examples of proteoglycans in the ECM.

A

Aggrecan
Perlecan
Decorin
CD44

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5
Q

Which cells primarily secrete ECM components?

A

ECM components are secreted locally by cells, primarily by fibroblasts.

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6
Q

What are the functions of GAGs and proteoglycans in the ECM?

A

Form highly hydrated gels embedding proteins.
Allow rapid diffusion of small molecules.
Resist compressive forces to maintain structural integrity.

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7
Q

What functions do ECM proteins serve?

A

Provide tensile strength: e.g., collagens, elastins.
Enable cell attachment: e.g., fibronectin, laminin.

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8
Q

How does matrix elasticity affect stem cell behavior?

A

Matrix elasticity directs stem cell lineage specification, influencing their differentiation based on the stiffness of the environment.

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9
Q

How does ECM stiffness influence cardiac cells?

A

Embryonic cardiomyocytes beat optimally on a matrix with heart-like elasticity.
Scar-like rigidity inhibits their beating and affects cardiac remodeling after injury.

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10
Q

What role does ECM stiffness play in axon growth in the developing brain?

A

Axons grow preferentially into areas of low ECM stiffness and avoid regions of high pressure.

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11
Q

Name diseases influenced by ECM stiffness.

A

Wound healing
Tumor metastasis: Matrix stiffness promotes cancer cell interactions with the endothelium.
CNS ageing: Niche stiffness affects central nervous system progenitor cells via the mechanosensitive ion channel PIEZO1.

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12
Q

What happens to a substrate when cells exert forces on it?

A

Cells deform the substrate by grabbing onto its surface.
Using a muscle contraction inhibitor (e.g., BDM) causes the substrate to relax and return to its original shape.

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13
Q

What is BDM, and what does it do?

A

BDM (2,3-butanedione monoxime) is a chemical inhibitor of skeletal and cardiac muscle contraction used to study cellular force dynamics.

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14
Q

What advantages do micropatterned PDMS hydrogels offer in cell adhesion studies?

A

Spatial control of adhesion sites.
Controllable rigidity of the adhesion surface.
Measurement of local forces exerted by adherent cells.

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15
Q

What is PDMS, and why is it used?

A

PDMS (polydimethylsiloxane) is a material used in hydrogels to study cellular mechanics due to its tunable elasticity and ability to mimic physiological environments.

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16
Q

Which ECM components are involved in outside-in adhesion, and how do they interact with cells?

A

Collagen and laminin interact with adhesion receptors.
Fibronectin (FN) binds to integrins, which are membrane receptors with cytoplasmic tails linking to intracellular proteins and actin fibers.

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17
Q

What is the structure of integrins?

A

Integrins are heterodimers formed by 8 beta chains and 18 alpha chains, creating 24 heterodimers.

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18
Q

What are some tissue-specific roles of integrins?

A

Leukocyte integrins help in adhesion to target cells.
Alpha5-beta1 integrin: Important in fibroblasts.
Alpha2b-beta3 integrin: A platelet receptor, crucial for wound healing.

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19
Q

Describe the structure of fibronectin.

A

Composed of two glycosylated chains (~250kDa, ~70nm) linked by disulfide bonds.
Exists in soluble and ECM forms.
Has alternatively spliced forms.

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20
Q

What are the functions of fibronectin?

A

Binds to integrins, bacteria, and other ECM components.
Plays roles in development, growth, wound healing, and cancer.

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21
Q

What is the effect of fibronectin gene knockout?

A

Lethal, indicating its critical role in cellular processes.

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22
Q

What are the two major states of fibronectin?

A

Compact (soluble) form: Found in blood circulation, binding to integrins is suppressed.
Extended (matrix) form: Forms fibrillar mesh in the ECM when activated.

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23
Q

What experimental methods show changes in fibronectin shape?

A

Analytical ultracentrifugation (AUC)
Electron microscopy (EM)

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24
Q

What is the primary binding site for cell attachment on fibronectin?

A

The Arg-Gly-Asp (RGD) tripeptide, located on the 10F3 domain.

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25
Q

Which FN modules mediate cell attachment to the matrix?

A

The 8th–10th F3 modules.

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26
Q

How does the FN synergy site influence cell spreading?

A

Spreading on 9F3-10F3 is as effective as on full-length fibronectin.
Spreading on 10F3 alone is only ~20%, indicating an additional interaction at 9F3.

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27
Q

How was the FN synergy site identified?

A

Through mutagenesis and spreading assays, showing that the PHSRN sequence on 9F3 is crucial.

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28
Q

What does the structure of 7F3-10F3 reveal about the synergy site?

A

The PHSRN sequence is on the same side as the RGD loop, enabling cooperative interaction with integrins.

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29
Q

What happens after cell attachment to fibronectin?

A

Cell flattening and spreading occur.
Cells can be adherent without fully spreading, depending on the interaction strength.

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30
Q

What is the primary integrin binding site on fibronectin?

A

The RGD (Arg-Gly-Asp) tripeptide on the 10F3 domain.

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31
Q

What additional site enhances fibronectin-integrin interaction?

A

The synergy site (PHSRN) on the 9F3 domain.

32
Q

Why is interdomain orientation important for integrin interaction?

A

RGD and PHSRN must be on the same side of fibronectin.
A 23° rotation between domains disrupts activity, showing fine-tuned orientation is critical.

33
Q

Are integrins typically active or inactive?

A

Integrins are mostly inactive in resting cells.

34
Q

What triggers integrin activation?

A

Signals such as cytokines can activate integrins.
Example: Activation of beta2 integrins in white blood cells enables adhesion to epithelial cells via ICAM binding.

35
Q

How does the conformational switch affect ligand binding?

A

Inactive conformation: No ligand binding (e.g., alphav-beta3 in Ca²⁺).
Active conformation: Ligand binding occurs when integrins are extended or activated (e.g., Mn²⁺ + activating mAb).

36
Q

What challenges arise when interpreting integrin conformations in vitro?

A

Differences in conformations between in vitro and in vivo may complicate ligand binding interpretations.
Questions arise regarding which conformation corresponds to active or inactive states and the kinetics of the switching process.

37
Q

What are some tools to study integrin conformations?

A

Protein engineering/mutations: Locking proteins in specific conformations using disulfide bridges.
Conformation-dependent antibodies: Detect epitopes specific to particular states.
Structural analysis: Solving structures of engineered proteins in locked conformations.

38
Q

What is the structure of the I-domain in integrins?

A

It adopts a Rossmann fold, with 6+ alpha-helices surrounding a central beta-sheet.
Contains the MIDAS (metal ion-dependent adhesion site) motif.

39
Q

How does the I-domain’s helix alpha7 regulate ligand binding?

A

Structural variations in helix alpha7 modulate ligand affinity:
Open conformation: High affinity for ligands (e.g., ICAM).
Closed conformation: Low or no ligand affinity.

40
Q

How were open/closed conformations linked to integrin activity?

A

Disulfide bridges were used to lock I-domains in specific conformations.
Open conformation: Showed ICAM binding and cell activation.
Closed conformation: Showed no ICAM binding or activation.

41
Q

What role do conformation-dependent antibodies play in studying integrins?

A

Antibodies like CBRM1/5 bind to specific epitopes on the open I-domain but block ligand binding.
They help correlate epitope accessibility with conformational states.

42
Q

What was observed in NMR studies of 9F3-10F3 analogues?

A

Minimal chemical shift differences: Local structure is the same.
Same backbone dynamics: Local flexibility is unchanged.
Interdomain orientation differed: ~23° tilt angle impacts activity.

43
Q

Why is RGD loop flexibility critical?

A

Flexibility allows the loop to interact effectively with integrins, crucial for function.

44
Q

What is the relationship between leg separation and ligand affinity in integrins?

A

Legs close, head down: Low ligand affinity.
Legs apart, head up: High ligand affinity.

45
Q

How does the RGD ligand affinity change between closed and open states of alpha5-beta1 integrins?

A

Closed state: Micromolar (μM) affinity.
Open state: Nanomolar (nM) affinity.

46
Q

How do ectodomain dynamics compare to intact integrins in cells?

A

Ectodomains are more dynamic, as transmembrane (TM) and tail domains pose barriers to extended conformations in intact integrins.

47
Q

What is the role of alpha2b-beta3 integrin in platelets?

A

Mediates platelet aggregation via binding to fibrinogen or von Willebrand factor, critical for wound healing.

48
Q

What structural changes occur in the I-like beta1 domain during ligand binding?

A

MIDAS expansion increases positive charge.
Rearrangement of metal ion coordination (e.g., D251 and M335) leads to high ligand affinity.

49
Q

What effect do constrained tail distances have on ectodomain conformation?

A

Constraining tails favors the bent conformation.
Coordinated movement of the I-like beta1 domain aligns with leg separation.

50
Q

How were jun/fos leucine zippers used in integrin studies?

A

Zippers were added to ectodomains to constrain tail distance.
Proteolytic cleavage of the clasp allowed release and observation of conformational changes.

51
Q

What is the role of the alpha2b tail in regulating integrin activity?

A

Acts as a negative regulator of beta3 integrins, keeping the integrin in an inactive state.

52
Q

What mutations lead to constitutive activation of alpha2b-beta3?

A

Removing the alpha2b tail or its GFFKR motif.
Mutations disrupting the R995-D723 salt bridge (e.g., R995A or D723A).

53
Q

What happens if charge reversal mutations are introduced to the salt bridge (e.g., R995D and D723R)?

A

The integrin remains inactive, showing the importance of specific charge interactions.

54
Q

What are key residues involved in ligand binding and integrin activation?

A

MIDAS residues (e.g., D251 and M335) for metal coordination.
Salt bridge residues (R995 and D723) for tail regulation.

55
Q

How is integrin activity linked to structural motifs?

A

Residue motifs in binding sites tune ligand interactions and activation states, allowing integrins to respond dynamically to environmental cues.

56
Q

What stabilizes the close conformation of integrin ectodomain legs?

A

Outer clamp: Glycine residues.
Inner clamp: R995-D723 salt bridge.

57
Q

How does Talin-1 K324 affect integrin TM interactions?

A

Talin-1 K324 competes with alpha2bR995 for beta3D723, disrupting the R995-D723 interaction. This dissociation reorients the beta3 helix and facilitates leg separation.

58
Q

What happens when tensile force is applied to low-affinity integrin states?

A

Tensile force alone: Results in closed, low-affinity state of the headpiece.
Tensile + lateral force: Leads to high-affinity state.

59
Q

What regulates integrin ligand affinity?

A

Heads down, legs together: Low affinity.
Heads up, legs crossed (closed): Low affinity.
Heads up, legs apart: High affinity.

60
Q

What changes occur in outside-in signaling?

A

MIDAS complementation by ligand.
Hybrid domain swing.
Legs apart for high ligand affinity.

61
Q

What initiates inside-out signaling?

A

Talin F3 binding to the beta3 tail (via the NPLY motif) dissociates the alpha2b-beta3 TM interaction, leading to leg separation and activation.

62
Q

How does the outside environment influence integrin clustering?

A

Spatially clustered ligands induce integrin clustering.
Bulky glycostructures on the cell surface sterically exclude integrins, trapping clusters.

63
Q

How does the inside environment promote clustering?

A

Transient focal contacts (FX): Circular structures, 100 nm size, ~1 min half-life.
FX mature into focal adhesions (FA): >100 nm size, ~20 min half-life.
FA can further develop into elongated structures.

64
Q

Which proteins promote integrin clustering?

A

Talin: Promotes clustering and activation.
Kindlin: Promotes clustering without activation.
Alpha-actinin: Reinforces clustering.

65
Q

What are the characteristics of focal adhesions?

A

Dynamic, with over 100 proteins identified.
Act as signaling hubs, interacting with cytokine and T-cell receptor signaling pathways.
Major sites for force sensing of the cellular environment.

66
Q

What are the layers of focal adhesions, and which proteins are found there?

A

Close to membrane (~40 nm): FAK and Paxillin.
Further inside (~40-80 nm): Vinculin and Zyxin.
Broad distribution (~80-100 nm): VASP and alpha-actinin.

67
Q

How is FAK activated from its auto-inhibited form?

A

Auto-inhibited form: FERM domain is associated with the kinase, leading to low tyrosine phosphorylation and inactivity.
Activated form: FERM and FAT domains dissociate from the kinase, enabling high tyrosine phosphorylation and activation.

68
Q

What are the key features of talin’s structure?

A

Size: 270 kDa, elongated (~60 nm) dimer.
Key domains:
FERM domain: Integrin (beta-tail) binding and PIP2 membrane association.
I/LWEQ domain: Actin binding.
Properties: Flexible and actin-cross-linking.
Function: Common step in beta-integrin-mediated signaling events.

69
Q

What are the two key ends of talin’s activity?

A

Integrin activation: FERM domain engages with integrin beta-tails at the plasma membrane.
Cytoskeletal engagement: Rod domain interacts with the actin cytoskeleton.

70
Q

What occurs in talin’s “on-state”?

A

FERM F2 domain targets the plasma membrane.
FERM F3 domain binds to beta3, releasing the self-interaction of integrin alpha-beta tails.

71
Q

How does talin respond to mechanical tension?

A

Talin undergoes reversible extension unfolding under tension.
Force range: 5-25 pN, with main extension length ~50 nm.
The R3 domain has the lowest unfolding resistance.

72
Q

What happens when talin is under mechanical stress?

A

Mechanical stress exposes buried sequence motifs (VBSs) for interaction with focal adhesion (FA) proteins like vinculin.
Stress transitions:
R3 unfolds, losing interaction with RIAM but gaining vinculin interaction (enhances actin cross-linking).
Further tension unfolds alpha-helical VBS motifs, weakening vinculin binding and focal adhesions.

73
Q

What are the molecular consequences of stretch activation?

A

Stretch activation can both create and destroy binding sites.
Example: Mutation in VBS of R3 alters tension resistance (Funfold) and cell spreading on stiff surfaces.

74
Q

What does talin’s mechanosensitivity imply for cellular processes?

A

Mechanosensitive code: Point mutations in VBS can shift cell behavior, e.g., adapting to stiffer surfaces.
Disease link: Mutations in talin affecting Funfold are associated with cancer.
Beyond FAs: Talin also contributes to post-synaptic density remodeling, impacting memory.

75
Q

How does talin integrate chemical signaling and mechanical sensing?

A

Controlled unfolding: Mechanism for force detection.
Applications: Diverse areas like disease progression and memory formation.

76
Q

What are the guiding principles of talin interactions?.

A

Localisation: Ensuring talin is in the right cellular area.
Unmasking: Exposing binding sites upon mechanical or chemical triggers.
Constructing “AND” conditions: Controlled generation of multiple weak interactions.

77
Q

Yay

A

You made it to the end! :)

BIOL3014 Molecular Cell Biology (4 decks)

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